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Anal Chem. 2014 Oct 21;86(20):10296-302. doi: 10.1021/ac5025842. Epub 2014 Oct 2.

Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

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1
The Institute of Cancer Research , 237 Fulham Road, London SW3 6JB, United Kingdom.

Abstract

Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

PMID:
25233145
PMCID:
PMC4206527
DOI:
10.1021/ac5025842
[Indexed for MEDLINE]
Free PMC Article

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